The long-term goal of this project is to determine how the key enzymes of nitrogen metabolism are regulated in Bacillus subtilis. Genetic and biochemical approaches will be used to study and reconstruct the regulation of the genes for glutamine synthetase and glutamate synthase and to test the hypothesis that this regulation is closely tied to the intracellular balance of carbon and nitrogen metabolites. For regulation of glutamine synthetase synthesis, the most important unanswered questions concern the respective roles of a negative regulatory protein, GlnR, and glutamine synthetase itself and the nature of the intracellular metabolic signal. Evidence for functional interaction between GlnR and glutamine synthetase will be sought. In addition, in vitro experiments will seek to demonstrate the mode of action of the regulatory protein(s) and the nature of the signal. For regulation of glutamate synthase synthesis, the major questions to be answered are the mechanism by which a positive regulatory protein, GltC, affects transcription and the nature of the metabolite(s) to which it responds. An attempt will be made to reconstruct regulation in an in vitro system using purified proteins. Since cells often perceive excess or limitation of particular nutrients relative to the availability of other nutrients, the hypothesis that certain nitrogen metabolism and carbon metabolism genes participate in a double feedback control will be evaluated. That is, it is anticipated that synthesis of glutamate and glutamine is sensitive not only to the availability of nitrogen-containing compounds but also to the availability of 2-ketoglutarate, the carbon skeleton of the amino acids in question. Conversely, synthesis of 2-ketoglutarate may respond to the availability of both carbon metabolites and nitrogen-containing compounds derived from it. This will be assessed in in vitro transcription experiments and by studying the properties of mutant strains blocked in various steps in the Krebs Cycle and glutamate/glutamine synthesis.